DAPM (4,4'-diaminodiphenylmethane) is proposed as a selective bile duct toxicant because DAPM causes morphological injury to biliary epithelial cells (BEC) of the intrahepatic bile ducts without effects on hepatocytes and rapidly impairs biliary functions, particularly glucose reabsorption from the biliary traCt, but does not alter hepatocellular transport functions. Little is known about the mechanisms by which DAPM and other cholangiodestructive agents damage bile ducts. By 3 hr after DAPM, BEC show ultrastructural alterations in mitochondria, loss of lumenal microvilli and dilation of Golgi Cisternae. Studies of liver mitochondria 3 hr after DAPM treatment demonstrate no alterations in enzyme activities, mitochondrial permeability characteristics, or histochemical staining patterns. The observation that BEC mitochondria are an early site of DAPM injury may explain the rapidity and severity of lesions caused by DAPM, and provides a unique opportunity to investigate mechanisms of bile duct toxicants. Our overall objective is to understand how and why DAPM causes early, selective injury to bile duct cells. The focus of this application will be on DAPM metabolites excreted in bile and their capacity to damage BEC. Our experimental approach will use the 25 mg/kg dose of DAPM which causes moderately injurious functional/structural alterations in BEC but does not alter bile flow or damage hepatocytes. Studies of metabolite excretion in bile and biliary function will be done in anesthetized rats infused with taurocholate intraduodenally to maintain bile flow. AIM 1 will clarify the effects of DAPM on tight junction permeability using paracellular markers and cytochemical ultrastructural methods. AIM 2 will characterize DAPM metabolites in vivo, particularly in bile, and will use isolated cells to assess DAPM cytotoxicity in vitro in hepatocytes versus BEC. AIM 3 will determine if inhibitors of conjugation reactions decrease Phase Il enzymes in both BEC and hepatocytes, identify possible proximate toxicants of DAPM by determining extent of injury in rats whose biliary excretion of DAPM has been modulated in vivo, and assess relative toxicities of suspect proximate toxicants by in vitro cytotoxicity assays. Our new data provide such strong evidence of the selective effect of DAPM on BEC, and particularly on BEC mitochondria, that a new AIM 4 proposes physiological and biochemical studies of mitochondrial function in isolated BEC treated with DAPM. The proposed research will contribute significantly to our understanding of the mechanisms of BEC injury and provide a new model system to ask questions about the role of BEC in bile formation.